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Biomedical subjects

G H Fey

Publications and source records attributed to G H Fey.

At least 37 records · Page 2Linked to original sources
PubMed1996

Receptors for interleukin (IL)-10 and IL-6-type cytokines use similar signaling mechanisms for inducing transcription through IL-6 response elements.

The cytoplasmic domain of the receptor for interleukin 10 (IL-10R) contains two box 3 sequence motifs that have been identified in the signal-transducing receptor subunits for IL-6-type cytokines and noted to be required for activating STAT3 and inducing transcription through IL-6-responsive elements. To determine whether the IL-10R has signaling functions similar to IL-6R in cells normally expressing these receptors, leukocytes of the B-, T-, and NK-cell lineages were treated with either cytokine. Both cytokines activated factors that bound to the sis-inducible element and included STAT1 and STAT3. The cell response to IL-10 characteristically differed from that to IL-2/IL-15, IL-4, and interferon gamma. The signaling capabilities of the IL-10R for activating specific STAT proteins and inducing gene transcription were defined by reconstitution of receptor functions in transfected tissue culture cells. COS-1 cells, co-expressing the human IL-10R and individual STAT proteins, confirmed a preference of the IL-10R for STAT3 and STAT1. Unlike many hematopoietin receptors, the IL-10R did not detectably activate STAT5. The IL-10R, together with reporter gene constructs containing different IL-6-responsive gene elements, reconstituted in hepatoma cells an induction of transcription by IL-10 that was comparable to that by IL-6. This regulation could not be appreciably modified by enhanced expression of STAT proteins. The similar actions of IL-10R and IL-6R on the induction of endogenous IL-6-responsive genes were demonstrated in hepatoma cells stably expressing the IL-10R. These receptor functions required the presence of the box 3 motifs, as shown by the analysis of the mouse IL-10R constructs containing progressively truncated cytoplasmic domains. The data demonstrate that the IL-10R, unlike other members of the interferon receptor family, is highly effective in recruiting the signaling pathways of IL-6-type cytokine receptors.

Acute-Phase Proteins↗
PubMed1996

A specific deletion in the breakpoint cluster region of the ALL-1 gene is associated with acute lymphoblastic T-cell leukemias.

A variety of chromosomal translocations to the ALL-1 gene are regularly observed in acute leukemias and are thought to play a key role in the leukemogenic process. Chimeric proteins are encoded by the breakpoint regions of the derivative chromosomes have been proposed to be the relevant oncogenic agents. In addition, internal duplications of the ALL-1 gene have been observed in patients with specific acute myeloid leukemias. Thus, it has been hypothesized that oncogenic variants of the ALL-1 protein may be generated by both chimerization and self-fusion, but the critical structural features endowing the altered proteins with their oncogenic potential are still unknown. Here a novel structural alteration of the ALL-1 gene was observed in three patients presenting with acute T-cell leukemia (ALL) without chromosomal translocations or self-fusions of the ALL-1 gene. These unrelated patients carried an internal deletion in one of the two alleles of the ALL-1 gene that eliminated parts of introns 7 and 8, together with exon 8. The deletion was found in 3 of 74 ALL patients, but not in acute myeloid leukemias, follicular lymphomas, or peripheral blood leukocytes from healthy donors. One ALL patient showed the deletion at diagnosis but no longer at remission or at 9 months after remission. These findings support the hypothesis that the ALL-1 protein may be converted to an oncogenic variant, not only by chimerization or self-fusion, but also by deletion of sequences coded by exon 8. They further suggest that these three different types of structural alterations of the ALL-1 protein may each cause a distinct disease phenotype. Alternatively spliced mRNA species omitting exon 8 were observed in 14 of 24 ALL patients without detectable macroscopic alterations of the ALL-1 gene and also in peripheral blood leukocytes from healthy donors.

Adolescent↗
PubMed1996

Exon/intron structure of the human ALL-1 (MLL) gene involved in translocations to chromosomal region 11q23 and acute leukaemias.

The acute lymphoblastic leukaemia (ALL)-1 gene on human chromosome 11q23 is the site of many locally clustered chromosomal alterations associated with several types of acute leukaemias, including deletions, partial duplications and translocations. Structurally variant proteins derived from the altered gene presumably cause the malignant transformation of early haemopoietic progenitor cells. According to previously published reports, the gene consisted of at least 21 exons spread over approximately 100 kb. In this report a set of genomic fragments was isolated that represent a total of 35 exons (exons 3-37) encompassing > 95% of the protein-coding region (except exons 1 and 2) and the 3'-non-translated region of the gene. The distances between these exons were determined and a detailed restriction map was produced. The majority of the exon/intron boundaries were sequenced and an intron-phase analysis was performed. The results form the basis for a greater understanding of the translocations and other structural alterations of the gene that conserve the open reading frame and thus produce presumably oncogenic variants of the ALL-1 protein.

Base Sequence↗
PubMed1996

Prolactin induction of the alpha 2-Macroglobulin gene in rat ovarian granulosa cells: stat 5 activation and binding to the interleukin-6 response element.

alpha 2-Macroglobulin (alpha 2M) is expressed at high levels in the corpus luteum of pregnant rats in response to PRL and rat placental lactogens. These studies document that PRL induction of alpha 2M mRNA occurs rapidly in granulosa cells differentiated to the preovulatory phenotype in the presence of FSH and steroid, is hormone specific [induced by PRL but not by LH or interleukin-6 (IL-6)], and involves tyrosine kinase activity. To analyze the cellular signaling events stimulated by PRL, transient transfections of granulosa cells and electrophoretic mobility shift assays were done using the IL-6 response element (IL-6RE) of the alpha 2M promoter. The IL-6RE consists of two gamma-activating like sequences (GAS) that bind the acute phase response factor (APRF/Stat 3) in rat liver and the mammary gland factor (MGF/Stat 5) from mammary tissue. By transfecting various alpha 2M promoter-luciferase reporter transgenes into the granulosa cell cultures, we show that the GAS-like sites together with the minimal -48 base pairs of the alpha 2M promoter can confer PRL inducibility to the luciferase reporter gene. These same GAS-like sequences of the alpha 2M promoter were used to analyze the DNA-binding activity of proteins in whole cell extracts prepared from differentiated granulosa cells exposed to PRL for 0.25, 0.5, 4, and 20 h. PRL rapidly stimulated the binding of a specific protein to labeled alpha 2M GAS-like oligonucleotide, and this PRL-induced binding activity was shown to contain Stat 5 but not Stat 1 or Stat 3, using specific antibodies in the electrophoretic mobility shift assays. Because both Stat 5 and Stat 3 proteins are present in the whole cell extracts of differentiated granulosa cells, PRL appears to activate detectable amounts of Stat 5 (and not Stat 3). Thus, the initial induction of the alpha 2M gene by PRL in differentiated rat granulosa cells involves, at least in part, the activation (tyrosine phosphorylation?) of Stat 5.

Animals↗
PubMed1995

Transcription factors Stat3 and Stat5b are present in rat liver nuclei late in an acute phase response and bind interleukin-6 response elements.

Proteins binding at the interleukin-6 response element of the rat alpha 2 macroglobulin gene were purified by a combination of chromatographic procedures including binding site-specific DNA-affinity chromatography as the principal step. Three polypeptides of 92, 91, and 86 kDa were enriched approximately 6,300-fold from nuclei of rat livers excised 12 h after the induction of an experimental acute phase response. Amino acid sequence analysis identified the 86- and 91-kDa species as two forms of the transcription factor Stat3 and the 92-kDa species as the factor Stat5b. This identification was confirmed by gel mobility shift-supershift experiments using specific antisera for Stat3 and Stat5. Unexpectedly, activated Stat5 was also detected in the nuclei of untreated control rats. cDNA clones representing Stat3 and two isoforms of Stat5b were isolated from a cDNA library prepared with mRNA from rat livers excised at the peak of an experimental acute phase response. Full-length Stat5b, predicted from cDNA, consisted of 786 amino acids, while the variant Stat5b delta 40C lacked 41 amino acids at the COOH terminus. The amino acid sequence of rat Stat5b showed 26.7% overall identity with rat Stat3, 87.3% with sheep Stat5a, 92.5% with murine Stat5a, and 98.7% with murine Stat5b.

Acute-Phase Proteins↗
PubMed1995

Expression and refolding of a high-affinity receptor binding domain from rat alpha 1-macroglobulin.

A recombinant version of the receptor binding domain of rat alpha 1-macroglobulin (RBDv) consisting of residues 1319-1474 has been expressed in E. coli. Competition experiments with 125I-labelled methylamine treated human alpha 2-macroglobulin reveal that the alpha 1-macroglobulin-RBDv exhibit the same high affinity for the alpha 2-macroglobulin receptor as the entire 40 kDa light chain from rat alpha 1-macroglobulin. It is therefore concluded, that all determinants for receptor interaction reside in the C-terminal approx. 150 residues of the alpha-macroglobulin subunit.

Amino Acid Sequence↗
PubMed1995

STAT3 and STAT5B are targets of two different signal pathways activated by hematopoietin receptors and control transcription via separate cytokine response elements.

Transient transfection of expression vectors for various members of the hematopoietin receptor family and STAT proteins into COS-1 cells indicated that each receptor was capable of stimulating the DNA binding activity of STAT1, STAT3, and STAT5B. However, gp130 preferentially activated STAT1 and STAT3. Activation of STAT5B differed from that of the other two in that the box 3 sequence motif in the cytoplasmic domain of gp130 was not required. Moreover, STAT5B and STAT3 enhanced gene transcription via separate regulatory elements. This study has identified two potential signal transduction pathways by which hematopoietin receptors, including the interleukin-6 receptor, control transcription of acute phase plasma protein genes in hepatic cells.

Acute-Phase Proteins↗
PubMed1995

Receptors for interleukin-3 (IL-3) and growth hormone mediate an IL-6-type transcriptional induction in the presence of JAK2 or STAT3.

To determine the specificity of signal transducer and activator of transcription (STAT) protein activation by box 3 motif-deficient hematopoietin receptors, expression vectors encoding the receptors for growth hormone, interleukin-3 (IL-3), and IL-4 were transiently transfected into COS-1 cells, together with expression vectors for Janus kinases (JAKs) and STAT proteins. Each receptor mediated a dose-dependent activation of STAT1 and STAT3, and for IL-3R and GHR this process was enhanced by JAK2. The data suggest that a box 3 motif in the cytoplasmic domain of the signal-transducing receptor to the JAK/STAT pathway. Transfection of the receptors, in combination with STAT3, into HepG2 cells reconstituted a cytokine-dependent stimulation of gene transcription through IL-6 response elements, providing evidence for a functional role of STAT3 in controlling gene expression.

Animals↗
PubMed1995

Isolation of two interleukin-6 response element binding proteins from acute phase rat livers.

Proteins binding at the IL-6 response element of the rat alpha 2 macroglobulin gene were purified by a combination of conventional chromatographic procedures and binding-site specific DNA affinity chromatography. The proteins were purified from the nuclei of rat livers, excised at the peak of an experimentally induced acute phase response. By this procedure three polypeptides of 92, 91 and 86 kD were enriched more than 6,000-fold. Partial proteolysis with lysyl endopeptidase and aminoacid sequence analysis of proteolytic peptides identified the 86 and 91 kD species as the transcription factor Stat3 and the 92 kD species as a Stat factor distinct from Stats 1 and 3. cDNA clones for Stats 1, 3 and this 92 kD factor were isolated from a cDNA library prepared with mRNA from acute phase rat livers. Parts of their DNA sequences were determined and the sequences of the purified peptides were found in these cDNA sequences. Thus, the identity of the factors as Stat3 and a Stat factor different from Stats 1 and 3 was confirmed. These results suggest, that APRF/Stat3 and p91/Stat1 are not the only factors mediating the effects of IL-6 on class 2 acute phase genes. The 92 kD Stat factor binding at the IL-6 RE probably also functions as a transcription factor in the cytokine-induced activation of the alpha 2M gene.

Acute-Phase Reaction↗
PubMed1995

Separate signaling mechanisms are involved in the control of STAT protein activation and gene regulation via the interleukin 6 response element by the box 3 motif of gp130.

The cytoplasmic receptor sequences required for the transcriptional control via the IL-6 response element (IL-6RE) and the hematopoietin receptor response element (HRRE) in hepatoma cells were defined by transient expression of wild-type and mutant granulocyte-colony stimulating factor receptor-gp130 chimeric receptors. gp130 generated two separate transcriptional signals, one of which was directed to IL-6RE and required an intact box 3 motif, and another, which was directed to HRRE and was box 3-independent. The activation of DNA-binding of STAT3 required the same gp130 domains as the IL-6RE response. A box 3-independent activation of STAT proteins was achieved by overexpression of the kinases JAK2 or TYK2. The increase in the DNA-binding activity of STAT proteins, however, did not result in a corresponding increase in transcription via either IL-6RE or HRRE. The data indicate that activation of the DNA-binding potential of STAT proteins via gp130 is not sufficient to achieve transcriptional up-regulation of specific target genes.

Animals↗
PubMed1995

The LIF response element of the alpha 2 macroglobulin gene confers LIF-induced transcriptional activation in embryonal stem cells.

Leukaemia Inhibitory Factor (LIF), an interleukin 6 (IL-6)-type cytokine, is an essential growth factor for murine embryonal stem cells. The LIF-receptor was known in these cells, but the cell-internal part of the signal cascade and the transcription factors through which LIF controls its growth-promoting target genes in embryonal stem cells, had not been identified. This study shows that the type II IL-6-response element of the rat alpha 2 macroglobulin (alpha 2M) gene, which mediates IL-6- and LIF-responses in hepatic cells, also functioned as a LIF-response element (LIF-RE) in ES1 embryonal stem cells and P19 embryonal carcinoma cells. It conferred transcriptional activation by LIF of transfected reporter constructs in these cells. A characteristic DNA-binding activity interacting with this LIF-RE was induced by treatment of these cells with LIF. The complex between this activity and the LIF-RE had identical electrophoretic mobility, sequence-specificity and kinetics of induction as the complex with the corresponding LIF-response factor (LIF-RF) from hepatic cells. The transcription factor STAT3 was part of this complex, as shown by its reactivity with anti-STAT3 antibodies. Withdrawal of LIF from ES1 cells caused the induction of differentiation and the disappearance of this DNA-binding activity. Simultaneously, the surface density of high-affinity LIF receptors was reduced approximately 10-fold.

Amino Acid Sequence↗
PubMed1995

Molecular analysis of the chromosomal breakpoint and fusion transcripts in the acute lymphoblastic SEM cell line with chromosomal translocation t(4;11).

The chromosomal breakpoint and fusion transcripts of the pre-B-leukaemia-derived SEM cell line carrying a reciprocal t(4;11)(q21;q23) translocation were analysed. The breakpoint from derivative chromosome der4 was cloned and sequenced. The crossover site was localized in intron 7 of the ALL-1 gene on chromosome 11q23 and in a large intron of the AF-4 (FEL) gene. RNA transcripts from both wild-type genes and both hybrid genes were detected by reverse transcriptase polymerase chain reaction (RT-PCR) assays. In addition, alternatively spliced mRNA species derived from the der4 chromosome were found. They were generated by using the exon 5' of the breakpoint on der4 as a common splice donor site and the 5' boundaries of exons 8 or 9 of the ALL-1 gene as alternative splice acceptor sites. The hypothesis is proposed that selective pressure operators to maintain the presence of both derivative chromosomes as important elements in the leukaemogenic process.

Base Sequence↗
PubMed1994

Cytokine-induced expression of leukemia inhibitory factor in renal mesangial cells.

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine, which shares many characteristics with interleukin-6 (IL-6). Recent observations indicate a role for LIF in inflammatory processes. To examine the potential involvement of LIF in the regulation of mesangial cell behavior, we studied LIF expression in early primary cultures of rat and human mesangial cells, as well as the response of mesangial cells to exogenous LIF. Growing or growth-arrested rat mesangial cells constitutively expressed very low levels of LIF mRNA, barely detectable by Northern blot analysis. Strong induction of LIF mRNA expression was caused by cytokines, such as interleukin-1 beta (5 ng/ml), tumor necrosis factor alpha (100 ng/ml) and PDGF (100 ng/ml), as well as LPS (200 ng/ml). The induction was transient with a peak after three to five hours. Dexamethasone (0.1 microM) almost completely inhibited the induction of LIF. Weak induction of LIF mRNA was observed after stimulation with basic fibroblast growth factor, endothelin and transforming growth factor beta. In combination with IL-1 beta, TGF beta showed synergistic effects on LIF induction. LIF itself or IL-6 had no effect on LIF mRNA expression. A similar induction pattern was observed for the expression of IL-6 mRNA. LIF protein was detected by specific ELISA in the supernatants of human mesangial cells stimulated by LPS or IL-1 beta. In addition, we found that mesangial cells not only express LIF but they are also target cells for LIF. Recombinant LIF effectively induced transient expression of the immediate early genes, c-fos, jun-B and Egr-1 in rat mesangial cells, with a maximum at 30 to 60 minutes. LIF was not mitogenic for mesangial cells. Our findings indicate that glomerular mesangial cells produce and react to LIF. As a cytokine with autocrine potential, LIF may play a physiological and/or pathophysiological role in the glomerulus, the exact nature and relevance of which remain to be explored.

Animals↗
PubMed1994

The acute lymphoblastic leukaemia cell line SEM with t(4;11) chromosomal rearrangement is biphenotypic and responsive to interleukin-7.

A cell line, designated SEM, was established from the peripheral blood of a 5-year-old girl in relapse with acute lymphoblastic leukaemia (ALL). Both the lymphoblasts of the patient and the cells of the cell line SEM showed the t(4;11) chromosomal rearrangement. The analysis of the immunophenotype of the SEM cell line revealed the B-cell differentiation antigens CD19, CD22 and CDw75 in the absence of CD20, CD24 and immunoglobulin expression. Besides B-lineage antigens, SEM cells were positive for the myeloid antigens CD13, CD15, CD33 and CDw65. Immunogenotypic analysis of SEM cells showed a monoclonal rearrangement of immunoglobulin heavy-chain (IgH). T-cell receptor (TCR) gamma and delta genes. Addition of interleukin (IL)-7 promoted the growth of the patient's lymphoblasts in culture and enhanced the proliferation of SEM cells. The SEM cells also express messenger RNA (mRNA) for the IL-7 receptor (IL-7R), but no evidence for autocrine production of IL-7 by the cell line was found. Addition of IL-4, tumour necrosis factor (TNF)-alpha, interferon (IFN)-alpha, or IFN-gamma resulted in a profound inhibition of SEM growth. Thus, these cytokines may have important growth regulatory activities for biphenotypic leukaemic ALL cells.

Antigens, CD↗